Poor fit on top

Hello. Ran this Ligand Ab. Analyte/Target- 28kD. HBS P+ as running buffer.. Sensorgram 1 having poor fit on top. (50nM). with KD 0.32 nM, high bulk RI took out top point, got 0.25 nM. Shown on sensorgram 2. Tried to set RI contant RI=0. Did not make a difference in the fitting. (sensorgram not shown). Is Sensorgram 2 acceptable? KD's look close. Kindly advise. Thanks.

Hi,
I think this is a nice and clear example of secondary binding events when you have a high analyte concentration. The 50 nM is about 156x the KD. The secondary binding is adding more analyte to the response distorting the 1:1 binding and the fitting tries to compensate by adding a buffer jump.
Leaving out the highest curve is improving the fitting. 10 nM = 40x KD of 0.25 nM.
For this high affinity binding I would suggest a longer association time if possible to get more curvature in the 10 nM concentration. We would accept the sensorgram 2.
Kind regards
Arnoud

Thanks Arnoud !